Method for efficient transfer of human blastocyst-derived stem cell (hbs cells) from a feeder-supported to a feeder-free culture system

ABSTRACT

A method for the transfer of human blastocyst-derived stem cells (hBS cells) to feeder-free culture system and propagation of the cells in such a feeder-free culture system, the method comprising the following steps of (a) transferring the balstocyst-derived stem cells from feeder to feeder free culture by mechanical treatment, (b) optionally, culturing the blastocyst-derived stem cells under feeder cell free growth conditions in a suitable growth medium and/or on a suitable support substrate, and (c) optionally passaging the blastocyst derived stem cell line every 3-10 days by enzymatic and/or mechanical treatment. The invention also relates to the application of hBS cells cultured under feeder free condition in medicine (e.g., myocardial regeneration) and screening and toxicity tests.

FIELD OF THE INVENTION

The present invention concerns a method for the transfer of humanblastocyst-derived stem cells (hBS cells) to feeder-free culture systemand propagation of the cells in such a feeder-free culture system. Theinvention also relates to the application of hBS cells cultured underfeeder free condition in myocardial regeneration.

BACKGROUND OF THE INVENTION

A stem cell is a cell type that has a unique capacity to renew itselfand to give rise to specialized or differentiated cells. Although mostcells of the body, such as heart cells or skin cells, are committed toconduct a specific function, a stem cell is uncommitted, until itreceives a signal to develop into a specialized cell type. What makesthe stem cells unique is their proliferative capacity, combined withtheir ability to become specialized. For years, researchers have focusedon finding ways to use stem cells to replace cells and tissues that aredamaged or diseased. So far, most research has focused on two types ofstem cells, embryonic and somatic stem cells. Embryonic stem cells arederived from the preimplanted fertilized oocyte, i.e. blastocyst,whereas the somatic stem cells are present in the adult organism, e.g.within the bone marrow, epidermis and intestine. Pluripotency tests haveshown that whereas the embryonic or blastocyst-derived stem cells(hereafter referred to as blastocyst-derived stem cells or (BS cells)can give rise to all cells in the organism, including the germ cells,somatic stem cells have a more limited repertoire in descendent celltypes.

In 1998, investigators were for the first time able to isolate hBS cellsfrom human fertilized oocytes and to grow them in culture see e.g. U.S.Pat. No. 5,843,780 and in U.S. Pat. No. 6,200,806.

The procedure used in the patent specifications mentioned above dependson the use of blastocysts with an intact zona pellucida. Furthermore,the method disclosed in these patents specifically use inner cell masscells that have been isolated by immunosurgery for plating on mouseembryonic feeder cells. This method has several drawbacks, for example,it is time consuming, technically difficult and results in low yields ofstem cells. Taken together, these drawbacks make it a costly method. Theso far few publications in the field illustrate the problems associatedwith establishing these stem cells from human blastocysts. As a resultvery few hBS cell lines are available.

Perhaps the most far-reaching potential application of hBS cells is thegeneration of cells and tissue that could be used for so-called celltherapies. Many diseases and disorders result from disruption ofcellular function or destruction of tissues of the body. Today, donatedorgans and tissues are often used to replace ailing or destroyed tissue.Unfortunately, the number of people suffering from disorders suitablefor treatment by these methods far outstrips the number of organsavailable for transplantation. The availability of hBS cells and theintense research on developing efficient methods for guiding these cellstowards different cell fates, e.g. insulin-producing β-cells,cardiomyocytes, and dopamine-producing neurons, holds growing promisefor future applications in cell-based treatment of degenerativediseases, such as diabetes, myocardial infarction and Parkinson's.

A significant challenge to the use of pluripotent stem cells for therapyis that they are traditionally cultured on a layer of feeder cells toprevent differentiation and to promote cell survival and proliferation.Without feeder cells in the culture environment, the stem cells willdie, or differentiate into a heterogeneous population of committedcells. Unfortunately, using feeder cells increases production costs,impairs scale-up, and produces mixed cell populations that require thepluripotent stem cells to be separated from feeder cell components.Furthermore, for therapeutic applications it will be of greatestimportance that the hBS cells are cultured without xenogenic tissuecontact, such as, e.g. feeder cells. Thus, there is a need fordeveloping methods for propagating human blastocyst-derived stem celllines without the use of feeder cells.

Other potential applications of hBS cells themselves and cellpopulations derived there from are found e. g. in the drug discoveryprocess in the pharmaceutical industry and in toxicity testings of allkinds of chemicals. Today, large-scale and high throughput screening ofdrug candidates usually relies on biochemical assays that provideinformation on compound binding affinity and specificity, but little orno information on function. Functional screening relies upon cell-basedscreens and usually uses organisms of poor clinical relevance such asbacteria or yeasts that can be produced cheaply and quickly at highvolume. Successive rounds of screening use model species of greaterclinical relevance, but these are more costly and the screening processis time consuming. Screening tools based on human primary cells orimmortalised cell types exist, but these cells are limited in supply orusefulness due to loss of vital functions as a result of in vitroculture and transformation. The access to undifferentiated hBS cells andhBS cells differentiated under engineered conditions and in the absenceof interfering feeder cells provides a new and unique capability toconduct human cell-based assays with high capacity, but withoutcompromising clinical relevance.

The following definitions and abbreviations are used herein

DEFINITIONS AND ABBREVIATIONS

As used herein, the term “blastocyst-derived stem cell” is denoted BScell, and the human form is termed “hBS cells”.

As used herein, the term “EF cells” means “embryonic fibroblast cells”.These cells could be derived from any mammal, such as mouse or human.

A “conditioned medium” is prepared by culturing EF cells or otherfibroblasts in a medium, and then harvesting and filtering the medium.

By the terms “feeder cells” or “feeders” are intended to mean cells ofone type that are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. The feedercells may optionally be from a different species as the cells they aresupporting. The feeder cells may typically be mitotically inactivatedwhen being co-cultured with other cells by irradiation or treatment withan anti-mitotic agent such as mitomycin c, to prevent them fromoutgrowing the cells they are supporting.

By the terms “feeder-free culture system”, “feeder cell free” or “feederfree” is intended to mean cultures or cell populations wherein less than10% of the total cells in the culture are feeder cells, such as, e.g.,less than 5%, less that 4%, less than 3%, less than 2%, less than 1%,less than 0.5%, less than 0.1% and less than 0.01%. It will berecognized that if a previous culture containing feeder cells is used asa source of hBS cells for the culture to which fresh feeders are notadded, there will be some feeder cells that survive the passage.However, after the passage the feeder cells will not proliferate, andonly a very small proportion will be viable in continuous cultures.

DESCRIPTION OF THE INVENTION

The inventors have established a novel method for the transfer of hBScells such as, e.g., a pluripotent human blastocyst-derived stem cellline from a fertilized oocyte to a feeder-free culture system and thenpropagating the cells in an undifferentiated state. The propagation isalso performed under-feeder free growth conditions.

According to many national laws in Europe and other countries, afertilized oocyte is not regarded as an embryo before implantation inthe uterus i.e. 10-14 days after fertilization. As the stem cell linesof the present invention are derived from a 4-5-days-old fertilizedoocyte, the stem cell lines should therefore not be regarded as anembryonic stem cell line. The right nomenclature of the stem cell linesof the present invention is blastocyst-derived stem cells. Furthermore,the stem cell lines of the present invention are not intended to use forhuman cloning and the creation of transgenic animals. The presentinvention does not concern a method to genetically modify the stem celllines.

The human blastocyst-derived cells suitable for use in a method of theinvention are derived from a group of cells called the inner cell mass,which is a part of the blastocyst. A blastocyst is a 4-5 days oldfertilized oocyte, which only upon implantation in the uterus candevelop to an embryo. Once removed from the blastocyst, the cells of theinner cell mass can be cultured into blastocyst-derived stem cells. Theblastocyst-derived stem cells are not intended to develop into embryos.

In a previous patent application published as WO 03/055992 (to the sameApplicant) a method for establishing hBS cells is described. Although itis contemplated that in the future it may be possible to establish suchcells without use of feeder cells, the current methods available usefeeder cells. However, future replacement therapies involving hBS cellsor tissues will require that the cells and tissues are produced withoutcontact with any animal (e.g. non-human) sources. Furthermore, the useof hBS cells also relies on the availability of routine large-scaleculturing protocols for undifferentiated hBS cells. The presentinvention addresses this issue by providing a suitable method fortransferring hBS cells from a feeder culture system to a feeder-freeculture system. hBS cells can be derived from the inner cell mass of thedeveloping blastocyst and maintained undifferentiated for an extendedperiod of passages while retaining stable karyotype and phenotype. hBScells have the capacity to differentiate into cells and tissues of allthree germ layers, both in vivo and in vitro, and are thus said to bepluritpotent. The unique properties of hBS cells suggest that they maysupply an almost unlimited source of cells for future replacementtherapies, functional genomics and proteomics as well as drug screening.

Mouse BS cells can be cultured without feeder cells if the medium issupplemented with leukaemia inhibitory factor (LIF). However, incultures of hBS cells, LIF does not have this effect. Today thederivation of hBS cell lines requires either human or mouse blastocystfibroblast feeders for co-culturing. Protocols for the transfer andpropagation of hBS cultures from feeder to feeder-free conditions havepreviously been described. These feeder-free culture protocols hadlimitations concerning scale-up properties, low success rate in theinitial transfer of the hBS cells from feeder culturing to feeder-freeconditions as well as generating a mixed population of undifferentiatedand differentiated hBS cells in the cultures.

The present invention provides an optimized method for transfer of hBScells to a feeder-free culture system, which method is advantageouscompared to the known methods in that the cells transferred are stablefor at least up to 10 passages. Studies by Richards et al. showed thatthe hBS cell lines could not be propagated in an undifferentiated statefor more than six passages on cell-free matrixes, including Matrigel™.However, the present inventors have found that the hBS cells were stablefor up to 35 passages on Matrigel™, still expressing the markers forundifferentiated hBS cells, even after a cycle of freezing/thawing andgrowth rates remained roughly comparable. Furthermore, a significantlyhigher number of surviving colonies were observed two days afterplating, when mechanical dissociation was compared with enzymaticdissociation. A critical step seems to bee the initial step for transferof the hBS cells to a feeder-free culture system. Accordingly, thepresent invention provides a method for transfer of hBS cells to afeeder-free culture system, wherein the hBS cells are mechanically cutfrom the feeder. In the examples herein, only the centre part of eachcolony was used, whereas in previous work by Xu et al., the wholecolonies were detached by enzymatic treatment with the risk ofcontaminating the cultures with feeder cells. Furthermore, the use ofenzymes, at the very delicate step of transferring the feeder culturedhBS cells to a feeder-free surface, may cause inactivation of importantsurface molecules involved in cell adhesion and growth. The majorcomponents in Matrigel™ are extracellular matrix proteins, like collagentype IV and laminin. Activation of the cell surface integrins uponbinding to extracellullar matrix proteins is believed to be a crucialstep for the regulation of cell adhesion, survival and proliferation.For example, Integrin alpha 1 has a unique role among the collagenreceptors in regulating both in vivo and in vitro cell proliferation incollagenous matrices. Laminin-specific receptors, possibly formed byIntegrin α6 and α1 which are highly expressed by hBS cells, may alsoplay a major role in the adhesion of hBS cell to the matrix surface.Thus, one possibility is that some of the important surface receptorsfor attachment or survival might be negatively affected by the roughinitial collagenase IV treatment before the cells have adapted to thenew surface.

In the examples herein different techniques for the transfer of hBScells to a feeder-free environment were investigated, either bymechanical or enzymatic dissociation, in regards to cell adhesion,survival rate and proliferation. Furthermore, method according to theinvention was developed in order to facilitate long-term propagation andlarge-scale production of homogenous populations of undifferentiated hBScells. The use of conventional cryopreservation techniques forfreezing/thawing of the hBS cells was also examined.

Transfer of hBS Cells to Feeder Free Propagation

Subsequent to dissection of the inner cell mass, the inner cell masscells are co-cultured with feeder cells to obtain a blastocyst-derivedstem (hBS) cell line. After obtaining the hBS cell line, the cell lineis optionally propagated to expand the amount of cells. Beforepropagation of the hBS cells in a feeder-free system, the hBS cells maybe transferred to a feeder-free system.

As mentioned herein and further demonstrated in the Examples a criticalfactor for the success in the propagation of the hBS cells is the methodby which the hBS cells is transferred from a feeder culture system to afeeder-free culture system. Accordingly, the hBS cells must betransferred to the feeder-free culture system by mechanical dissection,which may be performed by using a sterile sharpened glass capillary,with a 25 degree angle and a 200 or 300 micrometer lumen, designed forcutting, manipulation, and transfer of hBS colonies, or parts of hBScolonies. It is produced by Swemed Lab International AB, Billdal,Sweden.

As shown in the examples herein, mechanical dissociation resulted in amuch more efficient attachment of cells to the Matrigel™, a more rapidproliferation compared to the enzyme treated cultures, and the cellswere much more stable during passages. Accordingly, the method fortransferring the hBS cells according to the invention does not requireany enzymatic treatment. As seen in the examples herein, the cellscultured and proliferated under feeder-free conditions have a mitoticindex that was similar to that of cells grown under feeder conditions.

The propagation of the blastocyst-derived stem cell line comprisesculturing the stem cells under feeder cell free growth conditions, asculturing the hBS cells without feeder cells has a number of advantages,such as, e.g. there is no need for the ongoing production of feedercells, the production of hBS cells may be easier to scale up and thereis no risk of DNA transfer or other infection risks from the feedercells. If the medium is not correctly conditioned it may infect the newcell line.

Thus, the transfer and propagation step under feeder free conditions maycomprise the following steps of

-   -   a. transferring the blastocyst-derived stem cells from feeder to        feeder free culture by mechanical treatment.    -   b. optionally, culturing the blastocyst-derived stem cells under        feeder cell free growth conditions in a suitable growth medium        and/or on a suitable support substrate, and    -   c. optionally, passaging the blastocyst derived stem cell line        every 3-10 days by enzymatic and/or mechanical treatment.

In specific embodiments of the invention all steps i)-iii) are included.

Transfer of hBS Cells from a Feeder Culture System to a Feeder-FreeCulture System

The transfer step has been found to be a critical step as mentionedabove. Accordingly, the transfer should be done by means of mechanicallydissociation or mechanical dissection of the cells in the feeder culturesystem. This mechanical treatment may be done by means of any suitablecutting tool such as a tool having a sharpened end and a size that isappropriate for the cutting. The tool may be made of any suitablematerial such as, e.g., plastic or glass and an example of a suitabletool is a cutting tool that is a sterile sharpened glass capillary, witha 25 degree angle and a 200 or 300 micrometer lumen, designed forcutting, manipulation, and transfer of hBS cell colonies, or parts ofhBS cell colonies. It is produced by Swemed Lab International AB,Billdal, Sweden. In a specific embodiment the hBS cells to betransferred is a colony of hBS cells and pieces is cut from the centreof the colony and suspended in a suitable medium as cell clusters. Thecell clusters are dissociated mechanically one or more times e.g. untilthe cell clusters have a size that is at least 50% such as, e.g., at themost about 40%, at the most about 30%, at the most about 20%, at themost about 10% or at the most about 5% of that of the orginical colony.The size is e.g. determined as the diameter of the cluster or colony,respectively. In the examples herein is given suitable conditions forthe transfer process. These conditions may of course be varied withinappropriate limits, which is within the knowledge of a person skilled inthe art.

Culturing the Blastocyst Derived Stem Cells Under Feeder Cell FreeGrowth Conditions in a Suitable Growth Medium and/or on a SuitableSupport Substrate

The presence of a suitable growth medium, such as, e.g. a tissue culturemedium, and a support substrate, i.e. a growth support or coating, isvery important when growing cells under feeder free conditions. Whengrowing hBS cells on feeder cells, the feeder cells excrete varioussubstances that promote the proliferation and inhibit thedifferentiation of the hBS cells. When growing cells under feeder freeconditions such substances have to be supplemented to the growth mediumor coated on to the surfaces of the tissue culture wells, i.e. theinvention relates to a method, wherein the growth medium and/or thesupport substrate in step b) comprises substances that inhibitsdifferentiation and/or promotes survival and proliferation of theblastocyst-derived stem cells. Furthermore, the cells may need some kindof coating (support medium) to be able adhere to the surfaces of e.g.the tissue culture wells, that may be used for culturing the cells.

Such substances may be added to the media. Another way of preparing amedium comprising the suitable substances for promoting proliferationand inhibiting differentiation is to culture a first population of cellsin a medium, and then harvesting and filtering the medium (now denoted“conditioned medium”). The first population of cells may be cellsnormally used as feeder cells, such as e.g. mouse embryonic fibroblasts,human fibroblasts or cell lines derived from the same cells. Onesuitable medium for the culture of hBS cells is VitroHES™-medium(Vitrolife AB, Kungsbacka, Sweden) supplemented with 4 ng/ml humanrecombinant bFGF (basic fibroblast growth factor) or alternatively amedium termed “hBS-medium ” which may be comprised of; knockoutDulbecco's Modified Eagle's Medium, supplemented with 20% knockout Serumreplacement and the following constituents at their respective finalconcentrations: 50 units/ml penicillin, 50 g/ml streptomycin, 0.1 mMnon-essential amino acids, 2 mM L-glutamine, 100 M-mercaptoethanol, 4ng/ml human recombinant bFGF (basic fibroblast growth factor).

The conditioned medium (along with anything secreted into the medium bythe cells) may then be used to support the growth of a second populationof cells. A suitable medium for use according to the invention, is the“k-VitroHES™-medium” or “k-hBS-medium”, where a monolayer of mouse andhuman embryonic fibroblasts is mitomycin treated or irradiated and thenincubated with “VitroHES™-medium” or “hBS-Medium” for 24 hours. Thek-VitroHES™-medium or “k-BS-medium” may then be collected every day upto 3-7 times for mouse feeder and up to 3-7 times for human feeder fromthe same cells and sterile filtered to obtain the conditionedk-VitroHES™-medium or “k-hBS-medium”. The “k-VitroHES™-medium” and“k-hBS-medium” may subsequently be stored by freezing at about −20° C.or more.

In a specific example the growth medium in step b) may be cell-freeconditioned k-VitroHES™-medium or k-hBS-medium, produced by a culture offeeder cells as described in Example 3.

Another culture condition, which has been found to be favourable whengrowing hBS cell without feeder cells is the presence of a supportsubstrate, i.e. the invention relates to a method, wherein step a) isperformed on a support substrate. The support substrate is a surface orsurface treatment on e.g. tissue culture wells, which promotes theadhesion and growth of hBS cells in an undifferentiated state, i.e. thesupport substrate may comprise adhesion and proliferation promotingcomponents and components inhibiting differentiation, such as, e.g.,extra cellular matrix components such as, e.g., Matrigel™, human extracellular matrix (ECM) from placenta or laminin, or other components,such as, e.g. gelatine, polyornithine, fibronectin, agarose,poly-L-lysine or collagen type I.

Passaging the Blastocyst Derived Stem Cell Line Every 3-10 Days byEnzymatic and/or Mechanical Treatment

In a specific embodiment of the invention, the cells are passaged. Thenthe cells have to be passaged every 3-10 days, such as, e.g. about every3rd day, about every 4^(th) day, about every 5^(th) day, about every6^(th) day, about every 7^(th) day, about every 8^(th) day, about every9^(th) day and about every 10^(th) day. If the stem cell line iscultured longer than 10 days before passage, there is an increasedprobability that the cells undesirably will differentiate.

One way of dissociating the hBS cells is by enzymatic treatment or byusing a mild chelator such as EDTA. The enzymatic treatment may besupplemented by mechanical treatment to detach the cells from thesupport substrate and to complete the dissociation. The enzyme used maybe a collagenase, such as, e.g. collagenase IV. For the passaging theenzymatic treatment was found to be superior to mechanical treatment.

One of the other factors, which the present inventors have found may beimportant for the propagation of hBS cells under feeder free conditions,is the density of the cells when seeded onto the support substrate. Inorder to improve survival the cells may be plated at a density of80,000-200,000 cells/cm² depending on the cell lines used. The presentinventors have found that the hBS cells were stable for up to 60passages on Matrigel™, still expressing the markers for undifferentiatedhBS cells, even after a cycle of freezing/thawing and growth ratesremained roughly comparable.

Characterization

As described above, the present invention provides a method forpropagating hBS cells without feeder cells as described above, where thehBS cells maintain normal caryotype, stable proliferation rate andtelomerase activity. The cells are capable of proliferating in anundifferentiated state for more than 12 months when grown under feederfree growth conditions. The hBS cells, which are cultured underfeeder-free conditions, also expressed the markers associated withundifferentiated cells. Furthermore, the cells are able to developdifferentiated progeny from all three germ layers upon differentiationin vitro.

Methods Used to Study hBS Cell Degree of Differentiation andPluripotency

Immunohistochemistry

The hBS cells maintained in culture are routinely monitored regardingtheir state of differentiation. Cell surface markers used for monitoringthe undifferentiated hBS cells are SSEA-3, SSEA-4, TRA-1-60, TRA-1-81.Human BS cells are fixed in 4% PFA and subsequently permeabilized using0.5% Triton X-100. After washing and blocking with 10% dry milk thecells are incubated with the primary antibody. After extensive washesthe cell are incubated with the secondary antibody and the nuclei arevisualized by DAPI staining.

Alkaline phosphatase

The activity of alkaline phosphatase is determined using a commercialavailable kit following the instructions from the manufacturer (SigmaDiagnostics).

Oct-4 RT-PCR

The mRNA levels for the transcription factor Oct-4 is measured usingRT-PCR and gene specific primer sets (5′-CGTGAAGCTGGAGAAGGAGAAGCTG,5′-CAAGGGCCGCAGCTTACACATGTTC) and GAPDH as housekeeping gene(5′-ACCACAGTCCATGCCATCAC, 5′-TCCACCACCCTGTTGCTGTA).

Fluorescence In Situ Hybridization (FISH)

In one round of FISH one ore more chromosomes are being selected withchromosome specific probes. This technique allows numerical geneticaberrations to be detected, if present. For this analysis a commerciallyavailable kit was used, which contains probes for chromosome 13, 18, 21and the sex chromosomes (X and Y) (Vysis. Inc, Downers Grove, Ill.,USA). For each cell line at least 200 nuclei are being analyzed. Thecells are resuspended in Carnoy's fixative and dropped on positivelycharged glass slides.

Probe LSI 13/21 is mix with LSI hybridization buffer and added to theslide and covered with a cover slip. Probe CEP X/Y/18 is mixed with CEPhybridization buffer and added in the same way to another slide.Denaturing is performed at 70° C. for 5 min followed by hybridization at37° C. in a moist chamber for 14-20 h. Following a three step washingprocedure the nuclei are stained with DAPI II and the slides analyzed inan invert microscope equipped with appropriate filters and software(CytoVision, Applied Imaging).

Karyotyping

Karyotyping allows all chromosomes to be studied in a direct way and isvery informative, both numerical and larger structural aberrations canbe detected. In order to detect mosaicism, at least 30 karyotypes areneeded. However, this technique is both very time consuming andtechnically intricate. To improve the conditions for the assay themitotic index can be raised by colcemid, a synthetic analog to colchicinand a microtubule-destabilizing agent causing the cell to arrest inmetaphase, but still a large supply of cells are needed (6×10⁶cells/analysis). The cells are incubated in the presence of 0.1 μg/mlcolcemid for 1-2 h, and then washed with PBS and trypsinized. The cellsare collected by centrifugation at 1500 rpm for 10 min. The cells arefixed using ethanol and glacial acetic acid and the chromosomes arevisualized by using a modified Wrights staining.

Comparative Genomic Hybridization

Comparative genomic hybridization (CGH) is complementary to karyotyping.CGH gives a higher resolution of the chromosomes and is technically lesschallenging. Isolated DNA is nicktranslated in a mixture of DNA, A4,Texas red-dUTP/FITC 12-dUTP, and DNA polymerase 1. An agarose gelelectrophoresis is performed to control the size of resulting DNAfragments (600-2000 bp). Test and reference DNA is precipitated andresuspended in hybridization mixture containing formamide, dextranesulfate and SSC. Hybridization is performed on denatured glass slideswith metaphases for 3 days at 37° C. in a moist chamber. After extensivewashing one drop of antifade mounting mixture (vectashield, 0.1 μg/mlDAPI II) is added and the slides covered with cover slips. Slides aresubsequently evaluated under a microscope and using an image analysissystem.

Telomerase Activity

Since a high activity has been defined as a criterion for hBS cells thetelomerase activity is measured in the hBS cell lines. It is known thattelomerase activity successively decrease when the cell reaches a moredifferentiated state. Quantifying the activity must therefore be relatedto earlier passages and control samples, and can be used as a tool fordetecting differentiation. The method, Telomerase PCR ELISA kit (Roche)uses the internal activity of telomerase, amplifying the product bypolymerase chain reaction (PCR) and detecting it with an enzyme linkedimmunosorbent assay (ELISA). The assay is performed according to themanufacturer's instructions. The results from this assay show typicallya high telomerase activity (>1) for hBS cells.

Teratoma Formation in Immunodeficient Mice

One method to analyze if a human BS cell line has remained pluripotentis to xenograft the cells to immunodeficient mice in order to obtaintumors, teratomas. Various types of tissues found in the tumor shouldrepresent all three germlayers. Reports have showed various tissues intumors derived from xenografted immunodeficient mice, such as striatedmuscle, cartilage and bone (mesoderm) gut (endoderm), and neuralrosettes (ectoderm). Also, large portions of the tumors consist ofdisorganized tissue. Severe combined immunodeficient (SCID)-mice, astrain that lack B— and T-lymphocytes are used for analysis of teratomaformation. Human BS cells are surgically placed in either testis orunder the kidney capsule. In testis or kidney, hBS cells aretransplanted in the range of 10 000-100 000 cells. Ideally, 5-6 mice areused for each cell line at a time. Preliminary results show that femalemice are more post-operative stable than male mice and that xenograftinginto kidney is as effective in generating tumors as in testis. Thus, afemale SCID-mouse teratoma model is preferable. Tumors are usuallypalpable after approximate 1 month. The mice are sacrificed after 1-4months and tumors are dissected and fixed for either paraffin-orfreeze-sectioning. The tumor tissue is subsequently analyzed byimmunohistochemical methods. Specific markers for all three germlayersare used. The markers currently used are: human E-Cadherin fordistinction between mouse tissue and human tumour tissue, α-smoothmuscle actin (mesoderm), α-Fetoprotein (endoderm), and β-III-Tubulin(ectoderm). Additionally, hematoxylin-eosin staining is performed forgeneral morphology.

Cryopreservation and Thawing

As it appears from Example 6 herein, the hBS cells that have undergonepassaging can be cryopreserved and subsequently thawed. After thawingall cell lines survived and started to grow on Matrigel™ coated platesin similar patterns as before cryopreservation and thawing.

USE OF hBS CELLS OBTAINED ACCORDING TO THE INVENTION—CARDIO-RELATEDDISEASES

The hBS cells obtained by a method according to the invention may beused in medicine.

Coronary heart disease accounts for 50% of all cardiovascular deaths andnearly 40% of the incidence of heart failure. Sudden occlusion of amajor coronary artery and acute myocardial ischemia may lead to rapiddeath of myocytes and vascular structures. In the past, recovery ofcardiac function has been fully dependent on the growth of the remainingnon-infarcted portion of the ventricle. However, this is connected witha dilated myocardium, heart failure and death.

The only treatment currently available for replacing diseased myocardialtissue is organ transplantation. Because of the limited availability ofdonor hearts, however, relatively few potential recipients can benefitfrom heart transplantation. Even if the problems with cardiacavailability were overcome, the high costs involved in this procedureand the radical nature of the surgery would still limit organtransplantation to only those patients with end-stage diseased hearts.Thus, alternatives to organ transplantation are needed. In a specificaspect, the present invention concerns a method for the preparation ofhBS cells suitable for use as such an alternative.

Although prompt reperfusion within a narrow time window hassignificantly reduced early mortality from acute myocardial infarction,post-infarction heart failure resulting from ventricular remodeling isreaching epidemic proportions. Today, the only medical alternative forthese patients is to undergo heart transplantation. This is a veryexpensive treatment being afflicted with a high immediate risk, but alsowith severe post-operative complications. In addition, there is today agreat shortage of hearts for these types of transplantations. Instead,treatment with stem cells could be performed during acute surgery (e.g.open chest surgery), or at a later stage without surgery using e.g.balloon-catheter via the carotis artery or via systemic administration.Then time, suffering and risk for complications are reduced to aminimum. The advantage of stem cells versus organ transplantation isalso the unlimited access to material since stem cells can be propagatedindefinitely. Moreover, hBS cells are most certainly much lessimmunogenic active compared to adult hearts. Therefore, treatment ofthese patients with stem cells offers a time- and cost-effectivetreatment that also save a lot of human suffering. When stem cellstransplantation to damaged myocardium becomes a clinical reality, thistreatment has the potential to be the first of choice for millions ofpatients worldwide.

The hBS cells obtained by a method according to the invention may beused for the manufacture of a medicament for transplantation of hBScells into a mammal for the prevention or treatment of a disease suchas, e.g., a cardio-related disease including late myocardial infarction,chronic ischemic cardiomyopathy, idiopathic dilated cardiomyopathy,secondary cardiomyopathies such as, e.g. toxic, diabetic, pregnancy,amyloidosis, sarcoidosis, Fabry and haemochromatosis, end stagehypertrophic cardiomyopathy with LV dysfunction or heart failure,restrictive cardiomyopathy, end-stage hypertensive heart disease, acutemyocardial infarction, angina pectoris, fulminant myocarditis, AV-blockIII, specific forms of congenital heart diseases in children and adults,such as, e.g., noncompaction LV, atrial and ventricular septal defects,tetralogi Fallot and other similiar conditions, reconstruction of thevalves and heart failure secondary to valvular disease.

The medicament for transplantation of human blastocyst-derived stemcells may be designed to be administered into the myocardium or thecirculation of a mammal for the prevention or treatment of acardio-related disease. The medicament comprises undifferentiated hBScells or differentiated hBS cells dispersed in a pharmaceuticallyacceptable medium such as an aqueous medium. The medium may comprise oneor more additive selected from the group consisting of pH adjustingagents, stabilizers, preservatives, osmotic pressure adjusting agent,and physiologically acceptable salts; and/or one or more agents selectedfrom the group consisting of therapeutically active substances,prophylactically active substances, engraftment improving agents,viability improving agents, differentiation improving agent andimmunosuppressive agents.

Treatment of the Cultured Cells Before Administration

The following gives a description of a suitable method for treating thecells before administration. However, the description is included forillustrative purposes and is not intended to limit the invention in anyway.

The hBS cell colonies are dissociated in order to be of suitable sizefor transplantation and to give the cells optimal possibilities to enterand to be established in the host tissue. The colonies are partly orcompletely dissociated using mechanical or enzymatic treatment. Theenzymatic treatment may be performed with any suitable enzyme, such as,e.g. a solution of buffered collagenase or trypsin. Any suitablecollagenase may be used, such as, e.g., collagenase I, II, II, IV, V, IVetc. In the Examples is mentioned a specific example of collagenaseused. Also mechanical treatment with a pipette of the cell colonies inan EDTA-solution has been found efficient. After desired sizes of thecell-aggregates are achieved, the cell solution is centrifuged, washedand the pellet dissolved in an appropriate buffer for transplantation.

Administration

The hBS-cells are administered to animals as a sterile, bufferedsolution of cells, or cell-colony fragments by use of differentequipment and via different routes. A sufficient amount of cells areused. It is contemplated that about 10⁵-10⁸ cells are suitable. Thecells are aspirated into a sterile syringe and injected either directlyinto the animal or into a balloon-catheter that is placed in any of thecoronary vessels. The direct injection can be directed into the cardiactissue, to any of the cardiac cavities, or into the circulating blood.Cells can be administered with several injections at 1-3 different timepoints. During administration the general state of health of the animalis monitored. Moreover, the efficiency of cell transplantation in termsof leakage and cell loss is carefully followed. If needed, the animalreceives other pharmaceutical or immunosuppressing treatment. A definedcombination of cell transplantation and an agent improving the outcomeof the treatment could be beneficial.

If appropriate, the administration of the cells may be together with oneor more therapeutically or prophylactically active substance and/ortogether with one or more additives suitable for improving engraftmentand/or viability of the hBS cells and/or one or more immunosuppressiveagent. Moreover, they may be administered together with additivessuitable for improving differentiation of the cells. The administrationof these different agents may be before, concomitantly or after theadministration of the hBS cells.

The administration of the cells may be prophylactically, acute or aftersome time of progress of the disease.

The invention relates also relates to a kit comprising at least a firstand a second component in separate compartments. The components maycomprise an agent that improves the engraftment and viability of the hBScells, the hBS cells, one or more agents that improve differentiation ofthe hBS cells, and one or more pharmaceutical and/or immunosuppressingagents.

The kit may further comprise a second cell-type that improvesengraftment and survival of the hBS cells.

The kit may further comprise undissociated or dissociated differentiatedhuman BS-cell colonies.

As mentioned above, the invention also relates to the use ofdifferentiated cells such as, e.g., cardiomyocyte-like hBS cells. Belowfollows a description of the development of such differentiated cells.In the following the invention is described with specific reference tocardiomyocyte-like cells. However, other cells that differentiate fromhBS cells and that are suitable for use in the treatment ofcardio-related disease are intended to be included in the invention aswell.

Development of Differentiated Cells from hBS Cells

The hBS cell line obtained by a method as used in the present inventioncan be used for the preparation of differentiated cells. Therefore theinvention also relates to the differentiation of hBS cells into cardiactissue or cardiac related tissue, the cells itself and use of such cellsfor the preparation of medicaments for the treatment of cardio-relateddiseases, such as the ones mentioned above.

The hBS cells may be capable of forming cardiomyocyte-like structures,and the amount of these cells is generally higher than 10%, such as e.g.higher than 25%, or higher than 40%, or higher than 45%, or higher than50%.

The hBS derived stem cells may have the ability to differentiate intodifferentiated cells, which display the expression of cardiomyocytemarkers, including at least one of α-myosin heavy chain, α-actin,troponin I or troponin T, or one of the cardiomyocyte specific genes,including aGATA4, Mkx2.5, α-MHC, β-MHC or ANF.

Alternatively the hBS cells have the ability to differentiate intocardiomyocyte-like cells characterized by their organization intocontracting colonies, which are able to increase or decrease theirfrequency if α- or β-agonists or antagonists are administered to theculturing media.

The blastocyst-derived stem cells that are capable of being made intodifferentiated cells may be characterised with electron microscopy anddisplay a certain degree of myofibrillar organisation, consistent withearly-stage cardiomyocytes.

In another aspect, the invention relates to the use of a preparation ofdifferentiated cells derived from the blastocyst-derived stem cellsobtained by a method according to the invention for the manufacture of amedicament for the prevention or treatment of cardio-related diseases,such as e.g., myocardial infarction, cardiomyopathy, angina pectoris andheart failure secondary to valvular disease and the diseases mentionedabove.

A further object of the invention is to provide cells that may be usedfor the preparation of a medicament for treating and/or preventingdiseases that may be cured by “cell genesis”. By the term “cell genesis”is meant the generation of new cells such as cardiomyocytes, neurons,and/or different types of endothelium and vascular structures.

Treatment of the Differentiated Cells Before Administration

The differentiated cells may be treated in the same way as describedabove for the undifferentiated hBS cells.

Administration of Differentiated Cells

The differentiated cells may be administered in the same way as theundifferentiated hBS cells.

The invention also relates to a kit comprising at least a first and asecond component in separate compartments. The components may comprisean agent that improves the engraftment and viability of the hBS cells,the hBS cells, and one or more pharmaceutical and/or immunosuppressingagents.

The kit may further comprise a second cell-type that improvesengraftment and survival of the hBS cells.

The kit may further comprise undissociated or dissociatedundifferentiated human BS-cell colonies.

OTHER ASPECTS OF THE INVENTION

hBS cells can be used in high throughput screenings by combining highcapacity with improved clinical significance. The ability to preciselymodify the genome using gene targeting in hBS cells with or withoutdifferentiation of the genetically modified cells into various celltypes allows the application of this technology to the identification ofnovel therapeutically active substances through primary and secondaryscreening.

Accordingly, in other aspects the invention relates to the user of thehBS cells obtained by a method according to the invention defined for

i) the production of monoclonal antibodies,

ii) in vitro toxicity screening,

iii) in vitro screening of potential drug substances, or

iv) identification of potential drug substances.

The heart is the first functional organ in the human body. Therefore thecardio-like cells and/or the pathway to obtain these cells can be usedfor developmental toxicity testing by intervening (i.e. addingsubstances with potential toxic effect) and later monitoring thedevelopment in the test compared with a control group. Accordingly, theabove-mentioned aspects of the invention are of great importance.

Other embodiments of the invention appear from the appended claims. Thedetails and particulars described above and in the claims and relatingto the methods according to the invention apply mutatis mutandis to theother aspects of the invention.

The invention is further illustrated by the following figures:

FIGURE LEGENDS

FIG. 1. Blastocyst (before pronase treatment) from which human BS cellline 167 was established.

FIG. 2. Blastocyst (after pronase treatment) from which human BS cellline 167 was established.

FIG. 3. Blastocyst 167 two days after plating on embryonic mousefibroblasts.

FIG. 4. Human BS cells at passage 71 cultured on embryonic mousefibroblasts.

FIG. 5. A comparison chart of the two different techniques used for hEScell dissociation (Collagenase and Mechanical treatment), whenestablishing the cell lines on Matrigel™. The relative colony area (mm²)was compared between the two different dissociation techniques, on day 2and day 6 after transfer of the hES cells from mEF cultures toMatrigel™.

FIG. 6. Example of undifferentiated colony growth for cell line SA 167cultured on Matrigel™ for (a) 2 hours, (b) 10 hours, (c) 1 day, (d) 2days, (e) 3 days, (f) 4 days, (g) 5 days and (h) 6 days after seeding.

FIG. 7. Colony morphology of undifferentiated colonies of all four celllines (SA 002, AS 038, SA 121, SA 167) cultured on Matrigel™ on day 4after seeding.

FIG. 8. Examples of staining for alkaline phosphatase (AP) activity andfluorescent immunostaining performed on the undifferentiated cell lineSA167 cultured on Matrigel™ and after a cycle of freeze/thaw; (a) AP,(b) SSEA-1, (c) SSEA-3, (d) SSEA-4, (e) Tra-1-60, (f) Tra-1-81 staining.

FIG. 9. The relative telomerase activity (RTA), shown for Matrigel™cultures of cell line SA 121, AS 038, SA 167 and the negative control,in percentage of the positive control.

FIG. 10. Example of karyotypic analysis performed for cell line SA 121,cultured on Matrigel™ and after a cycle of freeze/thaw.

FIG. 11. Teratoma generated from Matrigel™ cultured cell line SA 002after injection under the renal capsule in immunodeficient SCID miceshowing; (a) teratoma overview, (b) ectodermal differentiation,neuroectoderm, (c) mesodermal differentiation, cartilage, and (d)endodermal differentiation, columnar epithelium with numerous gobletcells.

FIG. 12. RT-PCR analysis for Oct-4 expression performed for all fourcell lines (SA 002, AS 038, SA 121, SA 167) after establishment onMatrigel™ and after a cycle of freeze/thaw. The gel is 1.5% agarose,stained with ethidium bromide. (1) 100 bp DNA ladder, (2) cell line SA002, (3) cell line SA 121, (4) cell line SA 167, (5) cell line AS 038,and (6) negative control (water). Oct-4 PCR product is 247 bp.

FIG. 13. Percentage of cells in mitosis at day 3 of culture; acomparison between the hES cells cultured on mouse embryonic feederlayer (mEF) and on Matrigel™ (Cell line SA121).

FIG. 14. Flow-chart of the hBS cell line establishment, culture on mousefeeder, transfer to feeder-free culture, culture in the feeder-freesystem and injection of cultured cells into the myocard.

FIG. 15. Human BS cells from feeder-free culture in the rat myocard.Human cells are detected using an anti-human nuclear antigen antibody(green). Surrounding rat myocard cells are stained with the nuclearstain DAPI (blue).

REFERENCES

Gardner et al, Embryo culture systems, In Trounson, A. O., and Gardner,D. K. (eds), Handbook of in vitro fertilization, second edition. CRCPress, Boca Raton, pp. 205-264;

Thomson J A, Itskovitz-Eldor J, Shapiro S S et al. Embryonic stem celllines derived from human blastocysts. Science 1998;282:1145-1147.

Nico Heins, Mikael C. O. Englund, Cecilia Sjöblom, Ulf Dahl, AnnaTonning, Christina Berg, Anders Lindahl, Charles Hansson, and HenrikSemb; Derivation, Characterization, and Differentiation of HumanEmbryonic Stem Cells. Stem Cells, May 1, 2004, 22 (3)

Richards M, Fong C-Y, Chan W-K et al. Human feeders support prolongedundifferentiated growth of human inner cell masses and embryonic stemcells. Nat Biotechnol 2002;20:933-936.

Xu C, Inokuma M S, Denham J et al. Feeder-free growth ofundifferentiated human embryonic stem cells. Nat Biotechnol2001;19:971-974.

EXAMPLES Example 1

Establishment of an Essentially Pure Preparation of UndifferentiatedStem Cells from Spontaneously Hatched Blastocysts

Human blastocysts were derived from frozen or fresh human in vitrofertilized embryos. Spontaneously hatched blastocysts were put directlyon feeder cells (EF) in VitroHES™-medium supplemented with 4 ng/ml humanrecombinant bFGF (basic fibroblast growth factor) and 0.125 mg/mlhyaluronic acid. After plating the blastocysts on the EF cells, growthwas monitored and when the colony was large enough for manual passagingapproximately 1-2 weeks after plating the inner cell mass cells weredissected from other cell types and expanded by growth on new EF cells.

Example 2

Establishment of an Essentially Pure Preparation of UndifferentiatedStem Cells from Blastocysts with an Intact Zona Pellucida

For blastocysts with an intact zona pellucida (FIG. 1), a brief pronase(10 U/ml, Sigma) incubation in rS2 (ICM-2) medium (Vitrolife,Gothenburg, Sweden) was used to digest the zona (FIG. 2), after whichthe blastocyst was put directly on the EF cell layer in hBS mediumsupplemented with hyaluronic acid (0.125 mg/ml) (FIG. 3).

Example 3

Preparation of Conditioned VitroHES™-Medium (k-VitroHES™-Medium) forFeeder Free Cultures

To prepare mEF cells for conditioning of VitroHES™-medium, a confluentmonolayer of mEF cells (passage two) was Mitomycin C treated and seededin a concentration of 59 000 cells/cm² in a gelatin (0.1%; Sigma) coatedculture flask in Dulbecco's Modified Eagle Medium (D-MEM) supplementedwith 1% Penicillin/Streptomycin (PEST; 10000 U/ml), 10% Fetal BovineSerum (FBS) and 2 mM GLUTAMAX™-I Supplement (200 mM); all fromGibcoBRL/Invitrogen, Carlsbad, Calif., USA. After a 24 hour incubationperiod and one wash with PBS (GibcoBRL/Invitrogen), the medium wasdiscarded and replaced with VitroHES™-medium (0.28 ml/cm²)for a 24 hourconditioning period. The conditioned VitroHES™-medium(k-VitroHES™-medium) was collected every day up to three times from thesame mEF culture (in passage two) and sterile filtered by using a 0.2 μmlow protein binding filter (Sarstedt, Landskrona, Sweden). Thek-VitroHES™-medium was used either fresh or after freezing at −20° C.and supplemented with 4 ng/ml of bFGF (GibcoRL/Invitrogen) prior to use.The k-VitroHES™-medium may be used for up to one week if stored at +4°C. When stored at −20° C. for up to two months, no sign of reducedbioreactivity could be detected upon usage.

Example 4

Transferring of hBS Cell Lines to Feeder Free Growth Conditions

Initial hBS cell lines were maintained on Mitomycin C treated mousefeeders in 10-50 passages and cultured in VitroHES™-medium supplementedwith 4 ng/ml of human basic fibroblast growth factor (bFGF) (FIG. 4).

Two different techniques were evaluated for transferring of the hBScells from feeder culture to Matrigel™ coated plates, one withmechanical dissociation and one with collagenase treatment. The hBScells were cut in square pieces, which represented the middle of thecolony, by using a stem cell cutting tool (Swemed Lab AB, Billdal,Sweden), and carefully detached and transferred the cells to HBSSsolution. The stem cell tool is a sterile sharpened glass capillary,with a 25 degree angle and a 200 or 300 micrometer lumen, designed forcutting, manipulation, and transfer of hBS colonies, or parts of hBScolonies. It is produced by Swemed Lab International AB, Billdal,Sweden.

Enzymatic Treatment with Collagenase (for Comparison)

After washing in HBSS the cell clusters were transferred to aCollagenase IV solution (200 U/ml; Sigma) for enzymatic dissociation.The cells were incubated for 30 minutes at 37° C. and 5% CO₂. During theincubation period, repeated mechanical dissociations with a pipette wereperformed and the dissociation process monitored in an invertedmicroscope. After the incubation period the cell suspension was pelleted(400 G for 5 minutes) and washed once in KnockOut™ D-MEM(GibcoBRL/Invitrogen) before being resuspended in k-VitroHES medium.

MECHANICAL DISSOCIATION ACCORDING TO THE INVENTION

After washing in HBSS the cell clusters were carefully dissociatedmechanically by using a 1-ml automatic pipette. The dissociation processwas completed when the size of the cell clusters representedapproximately 1/10-1/20 of the original colonies (average of 20 000cells/original colony) corresponding to the size of cell aggregatesgenerated by Collagenase IV treatment, as described above After washingin HBSS the colonies were transferred to collagenase IV solution (200U/ml) to start the enzyme dissociation.

For the two different techniques, the cells were seeded into four wellseach and incubated at 37° C. in 5% CO₂. Each experiment was repeatedfour times, with the same amount of cells seeded each time. After twoand six days the colony size and number was calculated (FIG. 5).

Results of Example 3 and 4

To optimize the transferring of the hBS cultures from feeder tofeeder-free conditions, two different techniques were evaluated; onewith mechanical dissociation and one with enzymatic dissociation.Mechanical dissociation resulted in a more efficient attachment of cellsto the Matrigel™ and a more rapid proliferation compared to the enzymetreated cultures. A significantly higher number of surviving colonieswere observed two days after plating, when mechanical dissociation wascompared with enzymatic dissociation (FIG. 5). The total area of allcolonies generated on Matrigel™ after dissociation with the twodifferent techniques, respectively, was compared (P<0.001). Furthermore,six days after plating the total colony area in the mechanicallydissociated cultures were significantly increased compared with theenzymatically dissociated cultures (P=0.036). (FIG. 5).

Example 5

Culture and Passage of hBS Cells Cultured on Matrigel™

Four different cell lines SA 002, AS 038, SA 121 and SA 167 were used inall experiments. The cell lines were propagated on Matrigel™ for up to35 passages and the morphological appearance and other hBScharacteristics remained unaltered even after a cycle of freeze/thawing.All cultures consisted of well defined colonies of hBS cells withoutmorphological signs of differentiation. After about 3-6 days the cellswere passaged by taken away the medium and 1 ml of Collagenase IV (200U/ml) solution was added to each well and incubated for 15-20 minutes.To facilitate cell detachment from the surface mechanical dissociationwas performed followed by another 15 minutes of incubation. The cellswere then washed, resuspended in k-VitroHES™ medium and seeded at asplit ratio of 1:2 to 1:6 onto Matrigel™. The hBS cultures were passagedevery 5 to 6 days and the medium was changed every second to third day.

Result of Example 5

Observations were made that during passage of the hBS cells establishedon Matrigel™, enzyme treatment with Collagenase IV was needed to detachthe colonies from the surface. Enzymatic treatment during passage wasalso found to give an increased proliferation rate after seeding,compared to mechanical dissociation (FIG. 6, 7).

Example 6

Cryopreservation and Thawing of hBS Cells Cultured on Matrigel™

Four different cell lines SA 002, AS 038, SA 121 and SA 167 were treatedwith collagenase IV for 20-30 minutes to separate the cells from eachother before freezing. After centrifugation the cells were transferredto freezing medium, which contains k-VitroHES™-medium containing 10%DMSO, 30% serum replacement and 4 ng/ml of bFGF, in a concentration of 1million cells per ml freezing medium. The final cell suspension was amixture of both single cells and cell clusters. The cryotubes (0.5-1.0ml of cell suspension) were rapidly transferred to Nalgene freezingcontainer for storages in −80° C. over night or at least for 2 hoursbefore long-term storage in Liquid Nitrogen.

Thawing of the hBS Cells

k-VitroHES™-medium has to be prepared and preheated before thawing thecells by placing the cryotubes in 37° water bath until all of the cellsuspension was thawed. The cell suspension was transferred to thepreheated medium for 5 minutes before centrifugation (400 G in 5minutes). Matrigel™ thin layer coated (BD) wells were rehydrated byadding 1 ml of k-VitroHES™-medium to the wells and incubate 30 minutesin 37° C. The cell pellet was resuspended in k-VitroHES™-medium andtransferred to either 24- or 6-well Matrigel™ plates.

Example 7

Characterization of Feeder Free Cultured hBS Cells

All characterization experiments were performed after establishment onMatrigel™ and after a cycle of freeze/thaw.

Immunocytochemistry: The cultures were passaged as described above,seeded into 6- or 24-well Matrigel™ plates and cultured for six daysbefore performing the immunostaining. The cultures were washed in PBS,fixed with 4% formaldehyde (HistoLab, Gothenburg, Sweden) for 15 minutesat room temperature and then washed again three times in PBS. Themonoclonal primary antibodies used were directed against SSEA-1, −3 and−4 (1:200; Developmental Studies Hybridoma Bank, University of Iowa,Iowa City, Iowa), Tra-1-60, Tra-1-81 (1:200; Santa Cruz Biotechnology,Santa Cruz, Calif.), and polyclonal rabbit anti-Phospho-Histone H3(1:150; KeLab, Upstate). The primary antibodies were incubated overnight at 4° C. before being visualized using appropriate Cy3- orFITC-conjugated secondary antibodies (1:300; Jackson ImmunoResearchLaboratories, West Grove, Pa.). Cultures were also incubated with4′-6′Diamidino-2-phenylindole (DAPI; Sigma-Aldrich Sweden AB, Stockholm,Sweden), at a final concentration of 0.5 ug/mL for 5 minutes at roomtemperature, to visualize all the cell nuclei. The stained cultures wererinsed and mounted using DAKO fluorescent mounting medium (Dakopatts AB,Älvsjö, Sweden) and visualized in an inverted fluorescent microscope(Nikon Eclipse TE2000-U). Alkaline phosphatase (AP) staining of theMatrigel™ cultured hBS cells was carried out according to themanufacturer's instructions using a commercially available kit(Sigma-Aldrich).

Telomerase activity: Matrigel™ cultured hBS cells were harvested, lysedand telomerase activity analyzed by a PCR-based ELISA (Roche DiagnosticsGmbH, Mannheim, Germany) according to manufacturers instructions.

Karyotyping and FISH: The Matrigel™ propagated hBS cells designated forkaryotyping were incubated for 1 to 3 hours in colcemid (0.1 g/ml,Invitrogen, Carlsbad, Calif., USA), dissociated, fixated, mounted onglass slides and the chromosomes visualized by using a modified Wrightsstaining (#WS-32, Sigma). Preparation of metaphase plates was performedas previously described. For the fluorescence in situ hybridization(FISH) analysis, a commercially available kit (MultiVysion™ PBMulticolour Probe Panel; Vysis, Inc., Downers Grove, Ill.) containingprobes for chromosome 13, 18, 21 and the sex chromosomes (X and Y) wasused according to the manufacturer's instructions. Slides were analyzedusing an invert microscope equipped with appropriate filters andsoftware (CytoVision, Applied Imaging, Santa Clara, Calif.).

Teratomas: For the teratoma formation experiment, immunodeficient SCIDmice (C.B-17/lcrCrl-scidBR, Charles River Laboratories, Germany) wereused. Matrigel™ propagated hBS colonies were enzymatically detached fromthe surface by using Collagenase IV (200 U/ml), mechanically dissociatedinto small cell aggregates and approximately 50 000 to 100 000cells/organ were injected under the kidney capsule. Control animals weretreated with Cryo-PBS injections or with primary brain cells from alittermate. The animals were sacrificed eight weeks after injection andthe tumors were immediately fixed in a 4% solution of paraformaldehydeand paraffin embedded. For histological analysis the teratoma weresectioned to 8 μm and stained with Alcian Blue/Van Giesson.

RT-PCR analysis of Oct-4 expression: Total RNA was isolated from allfour Matrigel™ cultured hBS cell lines by using RNeasy Mini Kit (Qiagen)according the manufacturer's instructions. The cDNA was synthesized from1 μg of total RNA using AMV First Strand cDNA Synthesis Kit (Roche) andthe PCR reaction preformed by using Platinum Taq DNA Polymerase(Invitrogen). The PCR reaction included four initial step-down cycles,with two repeated cycles for every annealing temperature, withdenaturation for 15 seconds at 94° C., annealing temperature for 15seconds at 66° to 60° C. and extension for 30 seconds at 72° C. Thefollowing cycles included 35 repeats with annealing temperature at 58°C. The forward and reverse primer sequences for Oct-4 were previouslydescribed. -actin primers were used as internal controls (sense,5′-TGGCACCACACCTTCTACAATGAGC-3′; antisense,5′-GCACAGCTTCTCCTTAATGTC-ACGC-3′; 400 bp product). The PCR products weresize fractioned by gel electrophoresis using a 1.5% agarose gel. Humanliver was used as a positive control and water as negative control forthe PCR reaction.

Results of Example 6 and 7

Cell lines SA 002, AS 038, SA 121 and SA 167 were frozen and thawed byusing cryopreservation techniques to see if any changes in thecharacterization could be found. After thawing all four cell linessurvived and started to grow on Matrigel™ coated plates in similarpattern

Pluripotency and maintenance of the four different hBS cell lines infeeder-free conditions was demonstrated and compared to previous resultsfor feeder cultures of the respective cell lines. Thesecharacterizations were performed by examining the morphology, expressionof undifferentiated markers, telomerase activity, karyotype, anddifferentiation in vivo.

Immunocytochemistry: SSEA-1 expression was negative in all feeder-freecultured hBS cell lines as opposed to staining with antibodies againstSSEA-3, SSEA-4, TRA-1-60 and TRA 1-80 which show a clear positiveimmunoreaction as expected for pluripotent hBS cells. Further, the cellsdisplayed high levels of AP reactivity (FIG. 8) in all four Matrigel™propagated cell lines.

Telomerase activity: Analysis was preformed on three of the Matrigel™cultured hBS cell lines (AS 038, SA 121 and SA 167). The hBS cellscultured on Matrigel™ were found to have high levels of telomeraseactivity (FIG. 9).

Karyotyping and FISH: Karyotype analysis was preformed on two of theMatrigel™ cultured cell lines, AS 038 and SA 121. Three of three cellsfrom cell line AS 038 and ten of twelve cells from cell line SA 121 werefound to possess normal human 46, XY karyotype (FIG. 10). The remainingtwo cells from the SA 121 cell line expressed an abnormal karyotype of45, XY and 42, XY. Although, karyotypic changes seem to be normaloccurring events after prolonged culturing for both feeder andfeeder-free hBS cell cultures. In this study karyotypic analysis offeeder cultured hBS cells were comparable with results after Matrigel™propagation, suggesting that the hBS cell karyotype remains normal andstable under these feeder-free conditions. FISH analysis was performedon two of the Matrigel™ propagated cell lines (SA 121 (XY) and SA 167(XX)). Analysis was performed for chromosomes X, Y, 18, 13 and 21. Forboth cell lines tested at least 93% were normal. The results from theFISH analysis were comparable with results from feeder cultured hBS celllines.

Teratoma formation: Teratoma formation was performed for two Matrigel™cultured hBS cell lines, SA 167 and SA 002, and the results showed thatteratomas formed consisting of differentiated cells and tissuerepresentative from all three germ layers (endoderm, mesoderm andectoderm (FIG. 11), providing evidence that the Matrigel™ propagated hBScultures have retained their pluripotency.

Oct-4 expression: Oct-4 expression was high in all four cell linescultured on Matrigel™ (FIG. 12).

Example 8

Comparison of Mitotic Index of hBS Cells Cultured Under Feeder-FreeConditions on Matrigel™ Coated Plates Compared to hBS Cells Cultured onEmbryonic Mouse Feeder Cells

Cell line SA 121 was cultured in parallel under feeder-free conditionson Matrigel™ coated plates and on embryonic mouse feeder cells for 3days. The number of cells in mitosis was then quantified by nuclearimmunoreactivity for phosphorylated Histone H3. The mitotic index inboth cultures was calculated in order to compare the growth rate betweenfeeder-free and feeder cultured hBS cells.

Result of Example 8

The mitotic index was similar in cultures grown under feeder-free(Matrigel™) compared to feeder layer conditions (FIG. 13). The doublingtime for the feeder-free cultures were roughly the same (around 35hours) as for feeder propagated hES cells.

Example 9

Transplantation of Matrigel™ Cultured Cells to a Rat Heart

Human blastocyst-derived stem cell colonies prepared as described aboveare dissociated with a 0.5 ml collagenase-solution (Collagenase Type IV,lyophilized 179 units/mg, Gibco, Invitrogen Corporation, dissolved inHBSS to 200 U/ml), and transferred to a 15 ml tube (described in P10391PC/P10387). The tube is centrifuged at 400×g for 5 minutes. Thesupernatant (collagenase solution) is discarded and the pellet isdissolved in 5 ml pre-warmed sterile HBSS (37° C.). The tube iscentrifuged again at 400×g for 5 minutes. The supernatant is discardedand the pellet is dissolved in 25 μl pre-warmed sterile HBSS (37° C.).The cells are transferred to a sterile syringe and transported to theanimal surgery room.

The cells were administered to the anaesthetized and ventilated rateither via 1-2 direct myocardial-injections, via injection into the leftventricle, or via systemically intravenous administration.

More detailed, male Sprague-Dawley rats ˜200 g were used and MI wasinduced by direct cryo-injury using 3 mm probe. This procedure resultedin anterior MI engaging ˜15-20% of left ventricle (LV). The hBS cellswere transplanted by intramyocardial injection into the viablemyocardium close to the infracted area or via systemically intravenousadministration directly after cryo-injury. All animals were investigatedwith transthoracal echocardiography, continuous ECG and LVcatheterization 1 week after transplantation.

Post-mortem, the hearts were evaluated histologically for detection andcharacterization of hBS cells. There were no deaths in the rats treatedwith hBS cells and no arrhythmias were detected either. There were nosigns of abnormal tissue growth at the site of hBS cells engraftment.

The presence of human cells in the periinfarcted area was confirmed byhistological analysis. The heart was excised and the tissue surroundingthe injection area was dissected. This piece was frozen down in OCTsolution in a freeze-container (cryomold). The whole piece was thencryosectioned in 10 μm slices using a microtome. The slices were put onmicroslides (plus), which were put in the freezer. Just prior toimmunohistochemical analysis, the slides were thawed in roomtemperature, and around each heart-slice a circle was applied using anImmEdge Pen. The samples were fixed in 4% formaldehyde, washed 5 minwith PBS and 3×5 min in TBS. The slices were then incubated 30 min inroom temperature with blocking agent (goat serum), followed by 24 hincubation at 37° C. with primary antibody (mouse anti-human nucleus).The slides were then washed 3×5 min in TBS, followed by incubation for15 min in blocking solution as above. The slides were then incubated 2-3h at 37° C. with secondary antibody (goat anti-mouse) and washed 3×5 minin TBS. All slides were then DAPI-stained for 2 min and washed 5 min inPBS. Finally, the slides were mounted in fluorescence medium (S3023,DAKO), and human cells were identified using a fluorescencemicroscope.

Results of Example 9

hBS cells for treating cardio-related diseases by administration of hBScells were cultured in different ways. As appears from the examplesherein (FIG. 15), the outcome of administration of hBS cells isdependent of how the cells are cultured. If hBS cells were cultured onMEF, few or no cells have been found. If hBS cells were cultured onMatrigel™ and transplanted as in this example, a large amount of cellswere identified 24 h after transplantation using the technique describedabove. This suggests that the Matrigel™ culturing technique dramaticallyincrease the viability of the cells, or the possibility for the cells toestablish in the host tissue.

METHODS FOR ESTABLISHING hBS CELLS SUITABLE FOR USE IN A METHOD OF THEPRESENT INVENTION

In PCT application published as WO 03/055992 (to the same Applicatn) on10 Jul. 2003, i.e. after the priority date of the present invention, asuitable method for establishing hBS cells is described. In one aspectof the present invention, the cells employed are obtained by the methodclaimed in WO 03/055992, which is hereby incorporated by reference.

The method for establishing pluripotent human blastocyst-derived stemcells or cell line from a fertilized oocyte comprises the steps of

i) using a fertilized oocyte optionally, having a grade 1 or 2, toobtain a blastocyst, optionally having a grade A or B,

ii) co-culturing the blastocyst with feeder cells for establishing oneor more colonies of inner cell mass cells,

iii) isolating the inner cell mass cells by mechanical dissection,

iv) co-culturing of the inner cell mass cells with feeder cells toobtain a blastocyst-derived stem cell line.

v) optionally, propagation of the blastocyst-derived stem cell line.

As a starting material for this procedure, fertilized oocytes are used.The quality of the fertilized oocytes is of importance for the qualityof the resulting blastocysts. The human blastocysts in step i) of themethod may be derived from frozen or fresh human in vitro fertilizedoocytes. In the following is described a procedure for selectingsuitable oocytes for use in a method according WO 03/055992. It wasfound that an important success criterion for the present method is aproper selection of oocytes. Thus, if only grade 3 oocytes are applied,the probability of obtaining a hBS cell line fulfilling the generalrequirements (described below) is low.

Donated fresh fertilized oocytes: On day 0 the oocyte is aspirated inAsp-100 (Vitrolife), and fertilized on day 1 in IVF-50 (Vitrolife). Thefertilized oocyte is evaluated based on morphology and cell division onday 3. The following scale is used for fertilized oocyte evaluation:

Grade 1 fertilized oocyte: Even blastomers, no fragments

Grade 2 fertilized oocyte: <20% fragments

Grade 3 fertilized oocyte: >20% fragments

After evaluation on day 3, fertilized oocytes of grade 1 and 2 areeither implanted or frozen for storage. Fertilized oocytes of grade 3are transferred to ICM-2 (Vitrolife). The fertilized oocytes are furthercultured for 3-5 days (i.e. day 5-7 after fertilization). Theblastocysts are evaluated according to the following scale:

Grade A Blastocyst: Expanded with distinct inner cell mass (ICM) on day6

Grade B Blastocyst: Not expanded but otherwise like grade A

Grade C Blastocyst: No visible ICM

Donated frozen fertilized oocytes: At day 2 (after fertilization) thefertilized oocytes are frozen at the 4-cell stadium using Freeze-Kit(Vitrolife). Frozen fertilized oocytes are stored in liquid nitrogen.Informed consent is obtained from the donors before the 5-year limit haspassed. The fertilized oocytes are thawed using Thaw-Kit (Vitrolife),and the procedure described above is followed from day 2.

As described above, fresh fertilized oocytes are from grade 3 quality,and frozen fertilized oocytes are from grade 1 and 2. According to dataobtained by the establishment methods, the percentage of freshfertilized oocytes that develop into blastocysts is 19%, while 50% ofthe frozed fertilized oocytes develop into blastocysts. This means thatthe frozen fertilized oocytes are much better for obtaining blastocysts,probably due to the higher quality of the fertilized oocytes. 11% of theblastocysts derived from fresh fertilized oocytes develop into a stemcell line, while 15% of the blastocysts derived from frozen fertilizedoocytes develop into a stem cell line. In summary, of the fertilizedoocytes that were put into culture 2% of fresh fertilized oocytesdeveloped into a stem cell line, and 7% of frozen fertilized oocytesthat were put into culture developed into a stem cell line.

The culturing of the fertilized oocyte to the blastocyst-stage isperformed after procedures well-known in the art. Procedures forpreparing blastocysts may be found in Gardner et al, Embryo culturesystems, In Trounson, A. O., and Gardner, D. K. (eds), Handbook of invitro fertilization, second edition. CRC Press, Boca Raton, pp. 205-264;Gardner et al, Fertil Steril, 74, Suppl 3, 0-086; Gardner et al, HumReprod, 13, 3434,3440; Gardner et al, J Reprod Immunol, In press; andHooper et al, Biol Reprod, 62, Suppl 1, 249.

After establishment of blastocysts in step i) optionally derived fromfertilized oocytes having grade 1 or 2, the blastocysts having grade Aor B are co-cultured with feeder cells for establishing one or morecolonies of inner cell mass cells. After being plated onto feeder cells,their growth is monitored and when the colony is large enough for manualpassaging (approximately 1-2 weeks after plating), the cells may bedissected from other cell types and expanded by growth on new feedercells. The isolation of the inner cell mass cells is performed bymechanical dissection, which may be performed by using glass capillariesas a cutting tool. The detection of the inner cell mass cells is easilyperformed visually by microscopy and, according, it is not necessary touse any treatment of the oocytes with enzymes and/or antibodies toimpair or remove the trophectoderm.

Thus, the procedure of WO 03/055992 alleviates the need forimmunosurgery. By comparing the success-rate in using immunosurgeryversus the present method, which leaves the trophectoderm intact, it hasbeen observed that the much simpler, faster and non-traumatic procedureof avoiding immunosurgery is more efficient than immunosurgery. Theseprocedures make the preparation of stem cell lines, and thedifferentiation of these cell lines commercially feasible. From a totalof 122 blastocysts, 19 cell lines were established (15.5%). 42blastocysts were processed by immunosurgery and 6 of these resulted insuccessfully established cell lines (14%). Eighty blastocysts wereprocessed by the present method and 13 cell lines were established(16%).

Subsequent to dissection of the inner cell mass, the inner cell masscells are co-cultured with feeder cells to obtain a blastocyst-derivedstem (BS) cell line. After obtaining the hBS cell line, the cell line isoptionally propagated to expand the amount of cells. Thus, theblastocyst-derived stem cell line may be propagated e.g. by passage ofthe stem cell line every 4-5 days. If the stem cell line is culturedlonger than 4-5 days before passage, there is an increased probabilitythat the cells undesirably will differentiate.

A specific procedure of passaging the cells in a feeder culture systemis given in Esteblishment example 5 herein.

Human BS cell lines may be isolated either from spontaneously hatchedblastocysts or from expanded blastocysts with an intact zona pellucida.In the method described above the blastocyst in step i) is aspontaneously hatched blastocyst. For hatched blastocysts thetrophectoderm may be left intact. Either hatched blastocysts orblastocysts with a removed or partially removed zona pellucida may beput on inactivated feeder cells.

Zona pellucida of the blastocyst may be at least partially digested orchemically frilled prior to step ii) e.g. by treatment with one or moreacidic agents such as, e.g., ZD™-10 (Vitrolife, Gothenburg, Sweden), oneor more enzymes or mixture of enzymes such as pronase.

A brief pronase (Sigma) treatment of blastocysts with an intact zonapellucida results in the removal of the zona. Other types of proteaseswith the same or similar protease activity as pronase may also be used.The blastocysts can be plated onto said inactivated feeder cellsfollowing the pronase treatment.

In the establisment method step ii) and/or step iv) may be performed inan agent that improves the attachment of the blastocysts and/or ifrelevant the inner cell mass cells to the feeder cells. A suitablesubstance for this purpose is a hyaluronic acid.

A suitable medium for plating the blastocysts onto feeder cells can behBS-medium that may be complemented with hyaluronic acid, which seems topromote the attachment of the blastocysts on the feeder cells and growthof the inner cell mass. Hyaluronan (HA) is an importantglycosaminoglycan constituent of the extracellular matrix in joints. Itappears to exert its biological effects through binding interactionswith at least two cell surface receptors: CD44 and receptor forHA-mediated motility (RHAMM), and to proteins in the extracellularmatrix. The positive effects of HA during the establishment of hBS cellsmay be exerted through its interactions with the surfactant polar headsof phospholipids in the cell membrane, to thereby stabilize thesurfactant layer and thus lower the surface tension of the inner cellmass or blastocyst which may result in increased efficiency in bindingto the feeder cells. Alternatively, HA may bind to its receptors on theinner cell mass or blastocyst and/or to the feeder cells and exertbiological effects which positively influence the attachment and growthof the inner cell mass. According to this, other agents that may alterthe surface tension of fluids, or in other ways influence theinteraction between the blastocyst and feeder cells can also be used ininstead of hyaluronic acid.

In the method describe above culturing of the feeder cells is ofimportance for the establishment of the hBS cell line. The propagationof blastocyst-derived stem cell line may comprise passage of the feedercells at the most 3 times, such as e.g. at the most 2 times.

Suitable, feeder cells for use in a method of the invention arefibroblasts of e. g. embryonic or adult origin. In a method according tothe invention the feeder cells employed in steps ii) and iv) are thesame or different and originate from animal source such as e.g. anymammal including human, mouse, rat, monkey, hamster, frog, rabbit etc.Feeder cells from human or mouse species are preferred. Anotherimportant criterion for obtaining an hBS cell line fulfilling thegeneral requirements are the conditions under which the blastocysts arecultured. The blastocyst-derived stem cell line may accordingly bypropagated by culturing the stem cells with feeder cells of a density ofless than about 60,000 cells per cm², such as e.g. less than about55,000 cells per cm², or less than about 50,000 cells per cm². In aspecific embodiment, the propagation of blastocyst-derived stem cellline comprises culturing the stem cells with feeder cells of a densityof about 45,000 cells per cm². These values apply in those cases wheremouse feeder cells are used and it is contemplated that a suitabledensity can be found for other types of feeder cells as well. Based onthe findings of the present inventors, a person skilled in the art willbe able to find such suitable densities. The feeder cells may bemitotically inactivated in order to avoid unwanted growth of the feedercells.

The blastocyst-derived stem cell line obtained by the establishmentmethod described above maintains selfrenewal and pluripotency for asuitable period of time and, accordingly it is stable for a suitableperiod of time. In the present context the term “stable” is intended todenote proliferation capacity in an undifferentiated state for more than21 months when grown on mitotically inactivated embryonic feeder cells.

The stem cell line obtained by the establishment method described abovefulfils the general requirements. Thus, the cell line

-   -   i) exhibits proliferation capacity in an undifferentiated state        for more than 21 months when grown on mitotically inactivated        embryonic feeder cells,    -   ii) exhibits normal euploid chromosomal karyotype,    -   iii) maintains potential to develop into derivatives of all        types of germ layers both in vitro and in vivo,    -   iv) exhibits at least two of the following molecular markers        OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3,        SSEA4, TRA 1-60, TRA 1-81, and the protein core of a keratin        sulfate/chondroitin sulfate pericellular matrix proteinglycan        recognized by the monoclonal antibody GCTM-2,    -   v) does not exhibit molecular marker SSEA-1 or other        differentiation markers,    -   vi) retains its pluripotency and forms teratomas in vivo when        injected into immuno-compromised mice,    -   vii) is capable of differentiating.

The undifferentiated hBS cells obtained by the method described aboveare defined by the following criteria; they were isolated from humanpre-implantation fertilized oocytes, i.e. blastocysts, and exhibit aproliferation capacity in an undifferentiated state when grown onmitotically inactivated feeder cells; they exhibit a normal chromosomalkaryotype; they express typical markers for undifferentiated hBS cells,e.g. OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3,SSEA-4, TRA 1-60, TRA 1-81, and the protein core of a keratinsulfate/chondroitin sulfate pericellular matrix proteinglycan recognizedby the monoclonal antibody GCTM-2, and do not show any expression of thecarbohydrate epitope SSEA-1 or other differentiation markers.Furthermore, pluripotency tests in vitro and in vivo (teratomas)demonstrate differentiation into derivatives of all germ layers.

According to the above, the method proveds an essentially purepreparation of pluripotent human BS cells, which i) exhibitsproliferation capacity in an undifferentiated state for more than 21months when grown on mitotically inactivated embryonic feeder cells; ii)exhibits normal euploid chromosomal karyotype; iii) maintains potentialto develop into derivatives of all types of germ layers both in vitroand in vivo; iv) exhibits at least two of the following molecularmarkers OCT-4, alkaline phosphatase, the carbohydrate epitopes SSEA-3,SSEA-4, TRA 1-60, TRA 1-81, and the protein core of a keratinsulfate/chondroitin sulfate pericellular matrix proteinglycan recognizedby the monoclonal antibody GCTM-2 v) does not exhibit molecular markerSSEA-1 or other differentiation markers, and vi) retains itspluripotency and forms teratomas in vivo when injected intoimmuno-compromised mice, and vii) is capable of differentiating.Procedures for the detection of cell markers can be found in Gage, F.H., Science, 287:1433-1438 (2000) and are also described above.

The establishment method is described below in the following“establishment examples”. These examples are included herein forillustrative purposes only and are not intended to limit the scope ofthe invention in any way. The general methods described herein are wellknown to a person skilled in the art and all reagents and buffers arereadily available, either commercially or easily prepared according towell-established protocols in the hands of a person skilled in the art.All incubations were in 37° C., under a CO₂ atmosphere.

One suitable medium used is termed “BS-cell medium” or “BS-medium” andmay be comprised of; knockout Dulbecco's Modified Eagle's Medium,supplemented with 20% knockout Serum replacement and the followingconstituents at their respective final concentrations: 50 units/mlpenicillin, 50 g/ml streptomycin, 0.1 mM non-essential amino acids, 2 mML-glutamine, 100 M-mercaptoethanol, 4 ng/ml human recombinant bFGF(basic fibroblast growth factor).

Another suitable medium is “BS cell body medium”, this may be comprisedas follows; knockout Dulbecco's Modified Eagle's Medium, supplementedwith 20% knockout Serum replacement and the following constituents attheir respective final concentrations: 50 units/ml penicillin, 50 g/mlstreptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine and 100M-mercaptoethanol.

In the present context the term “stable” is intended to denoteproliferation capacity in an undifferentiated state for more than 21months when grown on mitotically inactivated embryonic feeder cells.

ESTABLISHMENT EXAMPLES Establishment Example 1

Establishment of an Essentially Pure Preparation of UndifferentiatedStem Cells from Spontaneously Hatched Blastocysts

Human blastocysts were derived from frozen or fresh human in vitrofertilized embryos. Spontaneously hatched blastocysts were put directlyon feeder cells (EF) in hBS cell medium (KNOCKOUT Dulbecco's ModifiedEagle's Medium, supplemented with 20% KNOCKOUT Serum replacement, andthe following constituents at the final concentrations: 50 units/mlpenicillin, 50 g/ml streptomycin, 0.1 mM non-essential amino acids, 2 mML-glutamine, 100 M -mercaptoethanol, 4 ng/ml human recombinant bFGF(basic fibroblast growth factor), supplemented with 0.125 mg/mlhyaluronic acid. After plating the blastocysts on the EF cells, growthwas monitored and when the colony was large enough for manual passagingapproximately 1-2 weeks after plating) the inner cell mass cells weredissected from other cell types and expanded by growth on new EF cells.

Establishment Example 2

Establishment of an Essentially Pure Preparation of UndifferentiatedStem Cells from Blastocysts with an Intact Zona Pellucida

For blastocysts with an intact zona pellucida, a brief pronase (10 U/ml,Sigma) incubation in rS2 (ICM-2) medium (Vitrolife, Gothenburg, Sweden)was used to digest the zona, after which the blastocyst was put directlyon the EF cell layer in hBS medium supplemented with hyaluronic acid(0.125 mg/ml).

Establishment Example 3

Histo-Chemical Staining for Alkaline Phosphatase

The cells were harvested for RT-PCR and histological (alkalinephosphatase) and immunocytochemical analysis (see below). RNA isolationand RT-PCR. Total cellular RNA was prepared using Rneasy Mini Kit(Qiagen) according to the manufacturer's recommendations. The cDNAsynthesis was carried out using AMV First Strand cDNA Synthesis Kit forRT-PCR (Roche) and PCR using Platinum Taq DNA Polymerase (Invitrogen).Histochemical staining for alkaline phosphatase was carried out usingcommercially available kit (Sigma) following the manufacturer'srecommendations.

Establishment Example 4

Preparation and Culturing of hBS Cell Line

Mouse embryonic fibroblasts feeder cells were cultivated on tissueculture dishes in EMFI-medium: DMEM (Dulbecco's Modified Eagle'sMedium), supplemented with 10% FCS (Fetal Calf Serum), 0.1M-mercaptoehanol, 50 units/ml penicillin, 50 g/ml streptomycin and 2 mML-glutamine (GibcoBRL). The feeder cells were mitotically inactivatedwith Mitomycin C (10 g/ml, 3 hrs). Human BS cell-colonies were expandedby manual dissection onto inactivated mouse embryonic fibroblasts feedercells.

Human BS cells were cultured on mitotically inactivated mouse embryonicfibroblasts feeder cells in tissue culture dishes with hBS-cell medium:knockout Dulbecco's Modified Eagle's Medium, supplemented with 20%knockout Serum replacement and the following constituents at theirrespective final concentrations: 50 units/ml penicillin, 50 g/mlstreptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 100M-mercaptoethanol, 4 ng/ml human recombinant bFGF (basic fibroblastgrowth factor). Seven days after passage the colonies were large enoughto generate hBS cell bodies.

hBS cell colonies were cut with glass capillaries into 0.4×0.4 mm piecesand plated on non-adherent bacterial culture dishes containing hBS cellbody medium: knockout Dulbecco's Modified Eagle's Medium, supplementedwith 20% knockout Serum replacement and the following constituents attheir respective final concentrations: 50 units/ml penicillin, 50 g/mlstreptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine and100M-mercaptoethanol. The hBS cell bodies, including cystic hBS cellbodies, formed over a 7-9-day period.

Establishment Example 5

Passage of hBS Cells

Before passage the hBS cells are photographed using a Nikon EclipseTE2000-U inverted microscope (10× objective) and a DXM 1200 digitalcamera. Colonies are passaged every 4-5 days. The colonies are bigenough to be passaged when they can be cut in pieces (0.1-0.3×0.1-0.3mm). The first time the cells are passaged, they have grown for 1-2weeks and can be cut in approximately four pieces.

The colonies are focused, one by one, in a stereo-microscope and cut ina checkered pattern according to the size above. Only the innerhomogeneous structure is passaged. Each square of the colony is removedwith the knife, aspirated into a capillary and placed on new feedercells (with the maximum age of 4 days). 10-16 squares are placed evenlyin every new IVF-dish. The dishes are left five to ten minutes so thecells can adhere to the new feeder and then placed in an incubator. ThehBS medium is changed three times a week. If the colonies are passaged,medium is changed twice that particular week. Normally a “half change”is made, which means that only half the medium is aspirated and replacedwith the equal amount of fresh, tempered medium. If necessary the entirevolume of medium can be changed.

Establishment Example 6

Vitrification of hBS Cells

Colonies with the appropriate undifferentiated morphology from the cellline are cut as for passage. 100-200 ml liquid nitrogen is sterilefiltered into a sufficient amount of cryotubes. Two solutions A and Bare prepared (A: 800 @I Cryo PBS with 1M Trehalose, 100 @I etylenglycole and 100 @I DMSO, B: 600 @I Cryo PBS with 1M Trehalose, 200 @Ietylen glycole and 200 @I DMSO) and the colonies are placed in A for 1minute and in B for 25 seconds. Closed straws are used to store thefrozen colonies. After the colonies have been transferred to a straw, itis immediately placed in a cryotube with sterile filtered nitrogen.

Establishment Example 7

Seeding of Embryonic Mouse Feeder (EMFi) Cells

The cells are inactivated with EMFi medium containing Mitomycin C byincubation at 37° C. for 3 hours. IVF-dishes are coated with gelatin.The medium is aspirated and the cells washed with PBS. PBS is replacedwith trypsin to detach the cells. After incubation, the trypsin activityis stopped with EMFi medium. The cells are then collected bycentrifugation, diluted 1:5 in EMFi medium, and counted in a Bürkerchamber. The cells are diluted to a final concentration of 170K cells/mlEMFi medium. The gelatin in the IVF-dishes is replaced with 1 ml cellsuspension and placed in an incubator. EMFi medium is changed the dayafter the seeding.

1. A method for transfer hBS cells into a feeder-free culture system,the method comprising the step of: a. transferring the hBS cells fromfeeder to feeder-free culture without enzymatic treatment but solely bymechanical treatment.
 2. The method of claim 1 further comprising thestep of b. culturing the blastocyst derived stem cells under feeder cellfree growth conditions in a suitable growth medium and/or on a suitablesupport substrate.
 3. The method of claim 1 further comprising the stepof: c. passaging the blastocyst derived stem cell line every 3-10 daysby mechanical treatment.
 4. The method of claim 1, wherein themechanical treatment is performed by dissociation or dissection of thehBS in the feeder culture system by means of a suitable cutting tool. 5.The method according to claim 4, wherein the hBS cells to be transferredis a colony of hBS cells, pieces is cut from the centre of the colonyand suspended in a suitable medium as cell clusters.
 6. The methodaccording to claim 4, wherein the cell clusters are dissociatedmechanically one or more times.
 7. The method according to claim 6,wherein the mechanical dissociation is performed until the cell clustershave a size that is at least 50% of that of the original colony.
 8. Themethod of claim 7, wherein the size is determined as the diameter of thecluster or colony, respectively.
 9. The method according to claim 2,wherein step b) is performed on a support substrate comprising acomponent, that inhibits differentiation and/or promotes survival andproliferation and/or adhesion of the blastocyst-derived stem cellsand/or an extra cellular matrix component.
 10. The method according toclaim 9, wherein the extra cellular matrix component is Matrigel™. 11.The method according to claim 2, wherein step b) comprises the use of agrowth medium comprising factors that inhibits differentiation and/orpromotes survival and proliferation of the hBS cells.
 12. A methodaccording to claim 11, wherein the growth medium is a cell-free medium,selected from the group consisting of k-hBS-medium, k-VitroHES™-mediumor VitroHES™-medium conditioned by previous exposure of feeder cells.13. A method according to claim 3, wherein step c) comprisesdissociation of the hBS cells by enzymatic treatment.
 14. A methodaccording to claim 13, wherein the enzyme is a collagenase.
 15. A methodaccording to claim 3, wherein step c) comprises dissociation of the hBScells by mechanical dissection.
 16. A method according to claim 3,wherein step c) comprises dissociation of the hBS cell by subjecting thehBS cells to an EDTA solution.
 17. A method according to claim 16.wherein the concentration of the EDTA solution is at the most about 100mM.
 18. A method according to claim 1, wherein the bBS cells: i) exhibitproliferation capacity in an undifferentiated state for more than 12months when grown under feeder free growth conditions, ii) exhibitnormal euploid chromosomal karyotype, iii) maintain potential to developinto derivatives of all types of germ layers both in vitro and in vivo,iv) exhibit at least two of the following markers OCT-4, alkalinephosphatase, the carbohydrate epitopes SSEA-3, SSEA-4, TRA 1-60, TRA1-81, and the protein core of a keratin sulfate/chondroitin sulfatepericellular matrix proteinglycan recognized by the monoclonal antibodyGCTM-2, v) do not exhibit marker SSEA-1 or other differentiationmarkers, vi) retain its pluripotency and forms teratomas in vivo wheninjected into immuno-compromised mice, and/or vii) are capable ofdifferentiate.
 19. A method according to claim 3, wherein passaged hBScells have the ability of further differentiating into cardio-like cellsrelating to cardiac tissue.
 20. A method to claim
 19. wherein thepassaged hBS cells have the ability of differentiating into cardio-likecells relating to cardiac tissue, which display the expression ofcardiomyocyte markers, including at least one of α-myosin heavy chainα-actin, troponin land troponin II or at least one of the cardiomyocytespecific genes, including GATA4, Mkx2.5, α-MHC, β-MHC or ANF.
 21. Amethod according to claim 19, wherein the cardio-like cells arecardiomyocyte-like cells characterized by their organization intocontracting colonies, which are able to increase or decrease theirfrequency when α or β agonists or antagonists are administered to theculturing media.
 22. A method according to claim 19, wherein the amountof cardio-like cells which are derived from the pluripotent human BScell line is higher than 25% of the total amount of the cells within theculture.
 23. The method according to claim 1, further comprising thestep of: c. passaging the blastocyst derived stem cell line every 3-10days by enzymatic and/or mechanical treatment.
 24. A medicine comprisingan effective concentration of the hBS cells obtained by a method ofclaim
 1. 25. A medicament for transplantation of hBS cells into a mammalfor the prevention or treatment of a disease comprising an effectiveconcentration of cells obtained by the method of claim
 1. 26. Amedicament for the prevention or treatment of cardio-related diseasescomprising an effective concentration of cells obtained by the method ofclaim
 1. 27. A medicament for transplantation of humanblastocyst-derived stem cells into the myocardium or the circulation ofa mammal for the prevention or treatment of a cardio-related diseasecomprising an effective concentration of cells obtained by the method ofclaim
 1. 28. A medicament according to claim 25 wherein the disease isselected from the group consisting of late myocardial infarction,chronic ischemic cardiomyopathy, idiopathic dilated cardiomyopathy,secondary cardiomyopathies such as toxic, diabetic, pregnancy,amyloidosis, sarcoidosis, Fabry and haemochromatosis, end stagehypertrophic cardiomyopathy with LV dysfunction or heart failure,restrictive cardiomyopathy, end-stage hypertensive heart disease, acutemyocardial infarction, angina pectoris, fulminant myocarditis, AV-blockIII, specific forms of congenital heart diseases in children and adults,such as noncompaction LV, atrial and ventricular septal defectstetralogi Fallot and other similar conditions, reconstruction of thevalves and heart failure secondary to valvular disease.
 29. A medicamentaccording to claim 25 comprising undifferentiated hBS cells ordifferentiated hBS cells dispersed in a pharmaceutically acceptablemedium.
 30. A medicament according to claim 29 wherein the medium is anaqueous medium.
 31. A medicament according to claim 29 comprising one ormore additives selected from the group consisting of pH adjustingagents, stabilizers, preservatives, osmotic pressure adjusting agents,and physiologically acceptable salts.
 32. A medicament according toclaim 25 comprising one or more agents selected from the groupconsisting of therapeutically active substances, prophylactically activesubstances, engraftment improving agents, viability improving agents,differentiation improving agent and immunosuppressive agents.
 33. Amethod for treating a cardio-related disease in a mammal comprisingtransplanting hBS cells obtained by the method of claim 1 intocardio-like cells in an effective amount to the mammal in need thereof.34. A kit comprising at least two of the following components inseparate compartments, an agent that improves the engraftment andviability of hBS cells, the hBS cells obtainable by the method of claim1, one or more agents that improve differentiation of hBS cell and oneor more pharmaceutical and/or immunosuppressive agents.
 35. A kitaccording to claim 34, further comprising a second cell-type thatimproves engraftment and survival of the hBS cells.
 36. A kit accordingto claim 34 further comprising undissoclated or dissociateddifferentiated hBS-cell colonies.
 37. A method for the production ofmonoclonal antibodies comprising utilizing the hBS cells obtained by themethod of claim
 1. 38. A method for in vitro toxicity testing comprisingutilizing the hBS cells obtained by the method of claim
 1. 39. A methodfor in vitro screening of potential drug substances comprising utilizingthe hBS cells obtained by the method of claim
 1. 40. A method foridentification of potential drug substances comprising utilizing the hBScells obtained by the method of claim 1.